Mass Spectrometry for Intact Mass Analysis of Antibodies and Antibody-Drug-Conjugates

26 Pages·2017·2.96 MB·English

by  Aran Paulus

Capillary Electrophoresis – Mass Spectrometry for Intact Mass
Analysis of Antibodies and Antibody-Drug-Conjugates
Aran Paulus, Chien-Hsun Chen, Andreas Krupke, Stephane Houel, Michael Wenz, and Daniel Lopez-Ferrer
45th International Symposium on High Performance Liquid Phase Separations and Related Techniques
Prague, Czech Republic, June 19, 2017
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Overview
• Introduction/Motivation
• Antibodies and Antibody-Drug-Conjugates and other “bodies”
• Workflows and Instrumentation
• CEMS of NIST mAb and NIST mAb derived ADC
• Top-Down Proteomics with CEMS
• Conclusions
• Acknowledgements

2
Paradigm Shifts in Pharmaceutical Industry
Small molecule drug Antibodies and ADC’s
First introduction to market: 1899 1986
Aspirin MW 180D Transtuzumab MW 145’531D
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Paradigm Shifts in Pharmaceutical Industry
Acetyl Salicylic Acid
~1320 - 1540 amino acids
2 heavy and 2 light chains
MW: ~150 000 D
4 Cys-Cys bonds
Minimum of 2 glyco sites
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Structures of mAb Derived Biopharmaceuticals
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Intact mAb and mAb Fragments
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NIST Monoclonal Antibody as Universally Available Standard with Charge Variants
NISTmAb, recombinant humanized IgG1κ
Number of amino acids: 1326
Disulfide bonds: 14(inter-chain), 2(intra-chain)
High-abundance PTM: N-terminal pyroglutamination
C-terminal lysine clipping
glycosylation
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Analytical Problems for mAb and ADC Comprehensive Characterization
• Primary Structure Assessment
• Charge Variants
• Separation (HPLC/CE) with MS (peptide mapping, middle down and top down MS)
• AA sequence and variants
• Intact mass (ESI-MS)
• Bottom-up peptide maps
• Middle-down (reduction, heavy/light chain, IDeS)
• Top down (ETD/ECD, UVPD, CID, etc)
• Glyco variants
• (released glycans, glycoprotein/peptides)
• Cystein-linked variants
• Higher Order Structures, Aggregates and mAb/Ag
• PK/Quantification
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Proteomics Workflows for mAb Characterization Bottom-Up, Middle-Down, Top-Down
•Proteomics workflows depend on combination of
• high resolving separation methods with
• sensitive and high resolution MS

•Bottom-up combines nanoLC with MS
•Misses PTM information

•Top down combines sample prep with MS
•No good LC method for proteins
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CEMS on Microfluidic Devices (ZipChip)
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